Sulfenic acid-reactive compounds and their methods of synthesis and use in detection or isolation of sulfenic acid-containing compounds

ABSTRACT

The present invention provides compounds of Formula I: 
     
       
         
         
             
             
         
       
     
     wherein: R 1  is a label (e.g., a detectable groups; an anti-tumor agent)s; L is present or absent and when present is a linking group; and x represents an integer from 1 to 10; or a pharmaceutically acceptable salt thereof. the compounds are useful for, among other things, identifying cysteine sulfenic acids in proteins and monitoring oxidative damage in proteins and cells.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/620,263, filed Oct. 19, 2004, the disclosure ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention concerns compounds useful for labeling, detectingand isolating sulfenic acid-containing proteins and other molecules, andmethods of making the same.

BACKGROUND OF THE INVENTION

Interest in the identification of cysteine sulfenic acids (R—SOH) inproteins by biochemists has grown substantially over the last decade astheir biological roles in redox regulation and catalysis within an arrayof cellular proteins have become better defined (1,2). In spite of theirimportance, only a limited set of tools to identify these species areavailable, and most of these are only applicable to in vitro studies ofpure, isolated proteins (3,4). Chemical modification of cysteinesulfenic acids by dimedone (5,5-dimethyl-1,3-cyclohexanedione) providesa useful way to “tag” these species with a specific, irreversiblealkylating agent, but the lack of any spectral signal or labelassociated with the dimedone requires that the detection of this tag beundertaken by mass spectrometry (4-7).

SUMMARY OF THE INVENTION

Sulfenic acids such as cysteine sulfenic acids in compounds such asproteins can be identified by their ability to form adducts withdimedone, but this reagent imparts no spectral or affinity tag to theadduct to readily provide for subsequent analyses of such taggedproteins. Because 1,3-cyclohexanedione showed at least equivalentreactivity toward cysteine sulfenic acids, this compound was used as thebasis for a synthetic procedure designed to add a functional group, analcohol, then link fluorophores or biotin through this sidechain. Theresulting compounds retain reactivity and specificity toward cysteinesulfenic acids in proteins, allowing for incorporation of thefluorescent or affinity label into the protein. Such compounds areuseful for labeling proteins or other molecules containing sulfenicacids. More particularly, such compounds are useful for labelingsulfenic acids in proteins for their detection and isolation fromcomplex protein mixtures, for quality control purposes, for detectionand isolation of proteins in the course of experimental procedures, as atherapeutic agent to modulate cell signaling and other biologicalpathways, to evaluate the “redox status” of cells through a“fingerprint” of protein oxidation status linked with establishing thedistinct patterns and extents of cellular changes due to particulartreatments, and for the industrial purification of proteins forsubsequent commercial purposes.

When conjugated to an antitumor agent, the compounds of the presentinvention are useful in the treatment of cancers such as breast, colon,lung, prostate, brain or liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Electrospray ionization mass spectrometry analysis of adductswith the sulfenic acid form of C165S AhpC. The mutant of the bacterialperoxidase AhpC (C165S) containing only the peroxidatic cysteine (Cys46)but not the resolving cysteine (Cys165) that participates in disulfidebond formation was treated with hydrogen peroxide to yield therelatively stabilized sulfenic acid form of the protein under anaerobicconditions, then incubated with 3-(2,4-dioxocyclohexyl)propyl7-methoxy-2-oxo-2H-chromen-3-ylcarbamate (DCP-MCC, compound 7) to yieldthe covalent protein adduct. Shown are the transformed data thatrepresent the relative abundance (shown as both the predicted andobserved masses) of four prominent species of C165S AhpC: the proteinwith the active site Cys46 in the thiol (20,600 amu), sulfinic acid(20,632 amu), or sulfonic acid (20,648 amu) states, or in a covalentcomplex with DCP-MCC (20,985 amu).

FIG. 2. Fluorescence spectra of 3-(2,4-dioxocyclohexyl)propyl2-(methylamino)benzoate (DCP-MAB, compound 6) before (solid) and after(dotted) reaction with cysteine sulfenic acid-containing protein.Labeled C165S AhpC was prepared as described in the Experimental Sectionwith the cysteine sulfenic acid form of the protein incubatedanaerobically with 5 mM of 6 for 60 min, then washed free of theunreacted reagent and buffer components into a final buffer of 50 mMTris-HCl at pH 8 using an Apollo concentrator. The absorbance at 347 nmof this sample was 0.14, and the fluorescence measurements were takenusing a semi-micro cuvette with the 0.4 mm cuvette width directed towardthe excitation beam and the 1.0 mm internal width directed toward theemission detector at 90°. Emission scans (370 to 500 nm) were collectedat an excitation wavelength of 353 or 357 nm, respectively, for the freeand protein-bound 6, and excitation scans (250 to 400 nm) were collectedat an emission wavelength of 440 or 430 nm, respectively. For subsequentexperiments to determine the rates of reaction of cysteine sulfenicacid-containing C165S AhpC with 6, excitation and emission wavelengthswere set at 357 and 464 nm, respectively (shown by arrows in thefigure).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

“Label” as used herein may be any suitable label or detectable orotherwise functional group, including but not limited to biotin, avidin,fluorophores, antigens (including proteins and peptides), antibodies,porphyrins, radioactive or stable isotopes, and (in some embodiments)anti-tumor or other therapeutic agents. “Anti-tumor agent” as usedherein may be any suitable anti-tumor agent, including but not limitedto vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics,actinomycin D, plicamycin, puromycin, gramicidin D, paclitaxel (TAXOL®,Bristol Myers Squibb), colchicine, cytochalasin B, emetine, maytansine,and amsacrine (or “mAMSA”). The vinca alkaloid class is described inGoodman and Gilman's The Pharmacological Basis of Therapeutics,1277-1280 (7th ed. 1985) (hereafter “Goodman and Gilman”). Exemplary ofvinca alkaloids are vincristine, vinblastine, and vindesine. Theepipodophyllotoxin class is described in Goodman and Gilman, supra at1280-1281. Exemplary of epipodophyllotoxins are etoposide, etoposideorthoquinone, and teniposide. The anthracycline antibiotic class isdescribed in Goodman and Gilman, supra at 1283-1285. Exemplary ofanthracycline antibiotics are daunorubicin, doxorubicin, mitoxantraone,and bisanthrene. Actinomycin D, also called Dactinomycin, is describedin Goodman and Gilman, supra at 1281-1283. Plicamycin, also calledmithramycin, is described in Goodman and Gilman, supra at 1287-1288.Additional chemotherapeutic agents include cisplatin (PLATINOL® BristolMyers Squibb); carboplatin (PARAPLATIN®, Bristol Myers Squibb);mitomycin (MUTAMYCIN,® Bristol Myers Squibb); altretamine (HEXALEN®,U.S. Bioscience, Inc.); cyclophosphamide (CYTOXAN®, Bristol MyersSquibb); lomustine [CCNU] (CEENU®, Bristol Myers Squibb); carmustine[BCNU] (BICNU®, Bristol Myers Squibb); irinotecan (CPT-11). Additionalexamples of therapeutic or antitumor agents that may be used to carryout the present invention include but are not limited to those describedin US Patent Application Publication No. 2005/0181977, the disclosure ofwhich is incorporated by reference herein in its entirety.

“Fluorophore” as used herein includes any suitable fluorophore,including but not limited to 2-(methylamino)benzoic acid(N-methylanthranilic acid), 7-methoxycoumarin-3-carbamate, orfluorescein.

“Linker” or “linking group” as used herein may be any suitable linkinggroup, including but not limited to groups comprising, consisting of orconsisting essentially of C, O, N, P and/or S (e.g., including H wherenecessary). In some embodiments the linker is not shown in genericstructures as the linker may be thought of as a portion of the label.

Subjects, tissues, cells, cell fractions, and proteins utilized to carryout the present invention may be of any suitable source includingmicrobial (including gram negative and gram positive bacteria, yeast,algae, fungi, protozoa, and viral, etc.), plant (including both monocotsand dicots) and animal (including mammalian, avian, reptile, andamphibian species, etc.). Mammalian subjects include both humans andother animal species treated for veterinary purposes (including but notlimited to monkeys, dogs, cats, cattle, horses, sheep, rats, mice,rabbits, goats, etc.)

The present invention provides compounds of formula I:

wherein:

R₁ is a label;

L is present or absent and when present is a linking group; and

x represents an integer from 1 to 10.

Compounds of formula I may be synthesized by: reacting the anion of3-ethoxy-2-cyclohexen-1-one with a protected halo lower alkyl alcohol[such as a 1-butyldimethylsilyl (tBDMS) protected halo lower alkylalcohol; most particularly 3-iodo-1-propanol] in a suitable organicsolvent (such as tetrahydrofuran) and hexamethylphosphoramide to form a6-lower alkyl-OtBDMS-3-ethoxy-2-cyclohexen-1-one solution, adding anammonium fluoride (such as tetrabutyl ammonium fluoride (TBAF)) to saidsolution to form an alcohol solution, adding a label to the alcoholsolution, and adding HCl to create the a ketone at the (C)3 position.

Methods of use. In general, the present invention provides a method ofdetecting a sulfenic acid containing target compound, comprising:contacting a compound as described herein (a compound of Formula I) witha target compound; and then detecting the presence or absence of bindingof said compound to said target compound; the presence of bindingindicating said target compound is a sulfenic acid containing compound.The method can be carried out in vitro or in vivo (e.g., where thecompound is administered to a subject as described below) in accordancewith known techniques or variations of such techniques that will beapparent to those skilled in the art given the present disclosure. Whencarried out in vitro the method can be performed on tissues, cells,cell-lysates or cell fractions, mixtures of compounds or individualcompounds that are subject to or susceptible to the formation ofsulfenic acids. The method can be utilized to determine whether asulfenic acid containing target compound is present or absent from asample suspected of containing the same. The method can be utilized tomonitor redox signaling pathways and networks in cells and tissues invitro. In specific embodiments the method can be utilized to identifycysteine sulfenic acids in a protein and/or monitor oxidative damage inproteins or cells. The method can be used to detect the formation ofsulfenic acids in compounds such as proteins, includingcysteine-containing proteins, when exposed to oxidants or oxidizingagents. The methods of the invention are useful for screening cells ortissue for exposure to environmental contaminants (particularlyoxidative contaminants) or oxidative stress for diagnostic and forensicapplications. For example, environmental toxins such as cigarette smoke(containing, e.g., benzo-a-pyrene) or automobile exhaust (with variousoxides of nitrogen or sulfur, or with carbon monoxide), orchemotherapeutic agents such as cisplatin, can cause oxidative damage tocells which can be assessed by assays for sulfenic acid generationconducted in the methods described herein. Further, manychemotherapeutic agents work through induction of apoptosis in cancercells that occurs through a redox signaling mechanism that can bediscovered, or potentially manipulated, through the above assays. Inaddition, ischemia-reperfusion injury during the transient blockage ofblood vessels (as in strokes or heart attacks) or as occurs withtransplanted organs also imparts oxidative damage, all of which can beassessed through imaging approaches or at the level of their moleculardetails through the forementioned assays. Still further, exposure toradiation, including ionizing radiation, can be monitored or detected bydetecting the formation of, or increased numbers of, sulfenic acids incells, tissues, or proteins exposed or thought to be exposed to suchradiation (e.g., a cell or tissue sample from an individual subject).

The methods of the invention can be implemented with any suitable assayformat, including but not limited to: (a) visualization of sulfenic acidamount and location in intact tissues or cells, in permeabilized cells,or in fixed tissues or cells; and (b) detection, isolation andidentification of molecules which are labeled based on their sulfenicacid content by applying the label top intact cells or tissues includingusing perfusion in anaesthetized animals), or to cell lysates, or tofractionated cell extracts, then isolating, detecting and identifyinglabeled proteins using chromatographic procedures, immunoaffinityprocedures and/or two-dimensional gel procedures for separation followedby mass spectrometry or Western blot analysis for identification oflabeled components.

A further aspect of the present invention is a method of labeling,detecting and/or isolating cysteine sulfenic acids in proteins, orpeptides containing cysteine sulfenic acids, or small moleculescontaining sulfenic acids, comprising: applying a spectral or affinitytag to the sulfenic acid-containing compound, wherein the spectral oraffinity tag is a compound as described herein (particularly compoundsof Formulas I, II, and III herein). The applying step may be carried outby adding said compound to a composition (e.g., a solution, suspension,emulsion, multi-phase mixture etc.) comprising said compound, alone orin combination with other compounds, to a said composition, and thenseparating protein or labeled small molecule from free compound, anddetecting the presence or absence of label on the protein in accordancewith known techniques suitable for the particular label employed.

A further aspect of the present invention is a method for monitoring ormodulating protein or cellular oxidative damage, potentially incombination with detection of protein phosphorylation or otherpost-translational modifications in a protein, of cysteine residues ofsaid protein, or in other molecules within a cell, comprising: reactinga spectral or affinity tag with a protein containing cysteine residues,wherein the spectral or affinity tag is a compound as described herein,and then measuring the activity of the labeled cysteine (e.g., bymeasuring the amount of label on the protein or cysteine in accordancewith known techniques). Such methods may be carried out in like manneras described above. Similar methods may be used to monitor oxidativedamage to cellular components in intact or permeabilized cells as well.Following incubation with the labeling reagent, unreacted reagent couldbe removed and cells imaged to quantify the level of incorporation ofthe label into cellular components. Additionally, cells subjected to thelabeling agent could be disrupted for the analysis or isolation oflabeled components as described above.

Linking groups. Linking groups that may be used to form covalentconjugates of two functional moieties are known in the art. See, e.g.,U.S. Pat. Nos. 6,420,377; 6,593,334; and 6,624,317. The specific linkinggroup employed will depend upon the particular synthetic method used tomake the covalent conjugate, as will be appreciated by those skilled inthe art. A suitable linking group will permit the joining of groups toprovide a metabolically stable conjugate. In general, the linking moietymay comprise an aliphatic, aromatic, or mixed aliphatic and aromaticgroup (e.g., alkyl, aryl, alkylaryl, etc.) and contain one or more aminoacids or hetero atoms such as N, O, S, etc. For example, the linkinggroup L may be a compound of the formula -L_(a)-L_(b)-, where L_(b) ispresent or absent and L_(a) and L_(b) are each independently selectedfrom the group consisting of:

wherein:

n is 0 to 6, a is 0 to 3 and b is 0 to 3; and R₂₅ is selected from thegroup consisting of alkylene, alkenyl, and arylenyl.

A particular example of the present invention is a compound of formulaII:

wherein x represents an integer from 1 to 10.

A further particular example of the present invention is a compound offormula III:

wherein x represents an integer from 1 to 10.

Other examples. Dimedone reacts with sulfenic acids because it has avery acidic and nucleophilic carbon (“an active methylene group”) thatis situated between two electron withdrawing groups (the two ketones ina 1, 3 orientation). So, other compounds in addition to 1, 3 diketonesare useful as sulfenic acid traps. Some examples of such other compoundsare shown in the scheme below. Particularly, other cyclic molecules likeMeldrum's acid or barbituric acid are also useful agents that are not 1,3 diketones (one a diester and the other a diamide), yet are also usefulas sulfenic acid traps. Linkages are carried out in accordance withtechniques that will be apparent to persons skilled in the art. Also,this reactivity is not limited to active methylene compounds in rings.Open chain molecules like diethyl malonate and malodinitrile are alsouseful in carrying out the present invention. There are numerouscombinations of functional groups that could be in the 1 and 3 positionsto yield an active methylene group (ketones, esters, nitriles,aldehydes, and nitro groups are non-limiting examples thereof). Ofinterest, each of these different compounds may have a different rateprofile with reaction with sulfenic acids. Generic structures are setforth at the bottom of the scheme below to exemplify minimal structuresneeded to react with a sulfenic acid (bearing in mind that somepotential compounds that could react with sulfenic acids (likemalodinitrile) would be eliminated as they do not have any good placesto add a label).

Formulations and administration. The term “active agent,” as usedherein, includes the pharmaceutically acceptable salts of the compoundsdescribed herein. Pharmaceutically acceptable salts are salts thatretain the desired biological activity of the parent compound and do notimpart undesired toxicological effects. Examples of such salts are (a)acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid and the like; and salts formed with organic acids such as,for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonicacid, and the like; and (b) salts formed from elemental anions such aschlorine, bromine, and iodine.

Active agents used to prepare compositions for the present invention mayalternatively be in the form of a pharmaceutically acceptable free baseof active agent. Because the free base of the compound is less solublethan the salt, free base compositions are employed to provide moresustained release of active agent to the target area. Active agentpresent in the target area which has not gone into solution is notavailable to induce a physiological response, but serves as a depot ofbioavailable drug which gradually goes into solution.

The compounds of the present invention are useful as pharmaceuticallyactive agents and may be utilized in bulk form. More preferably,however, these compounds are formulated into pharmaceutical formulationsfor administration. Any of a number of suitable pharmaceuticalformulations may be utilized as a vehicle for the administration of thecompounds of the present invention.

The compounds of the present invention may be formulated foradministration for the treatment of a variety of conditions. In themanufacture of a pharmaceutical formulation according to the invention,the compounds of the present invention and the physiologicallyacceptable salts thereof, or the acid derivatives of either (hereinafterreferred to as the “active compound”) are typically admixed with, interalia, an acceptable carrier. The carrier must, of course, be acceptablein the sense of being compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier maybe a solid or a liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a tablet, which maycontain from 0.5% to 95% by weight of the active compound. One or moreof each of the active compounds may be incorporated in the formulationsof the invention, which may be prepared by any of the well-knowntechniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

In general, the formulations of the invention are prepared by uniformlyand intimately admixing the active compound with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture. For example, a tablet may be prepared by compressingor molding a powder or granules containing the active compound,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the compound in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets may be made by molding, in a suitable machine,the powdered compound moistened with an inert liquid binder.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compound, which preparations are preferably isotonic with theblood of the intended recipient. These preparations may be administeredby means of subcutaneous, intravenous, intramuscular, or intradermalinjection. Such preparations may conveniently be prepared by admixingthe compound with water or a glycine buffer and rendering the resultingsolution sterile and isotonic with the blood.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3:318 (1986))and typically take the form of an optionally buffered aqueous solutionof the active compound. Suitable formulations comprise citrate orbis\tris buffer (pH 6) or ethanol/water and contain from 0.01 to 0.2 Mactive ingredient.

Antitumor/anticancer or other therapeutic uses. When labeled with anantitumor or other pharmaceutical agent, compounds of the presentinvention are useful in the treatment of cancer or other diseases asnoted above. For such purposes the compounds may be provided as apharmaceutical formulation in a suitable pharmaceutical carrier, forexample an aqueous carrier such as sterile pyrogen-free water or salinesolution. The pharmaceutical formulation may be administered to asubject (e.g., a human subject, or other mammalian subject such as adog, cat, or monkey for veterinary purposes) afflicted with a cancer asnoted above by any suitable means, typically parenterally (e.g.,intraveneous, subcutaneous, intraperitoneal injection, etc.) in anysuitable amount (from about 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg or 0.3mg/kg; to about 0.3 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 10.0 mg/kg, ormore).

The present invention is explained in greater detail in the non-limitingExperimental section set forth below.

EXPERIMENTAL

In order to provide a new tool for identifying and isolating sulfenicacid-containing proteins and peptides, we designed and synthesized afunctionalized derivative of the dimedone-like compound1,3-cyclohexadione (3 in Scheme 1), followed by linkage of the alcoholfunction to two different fluorophore groups, isatoic acid and7-methoxycoumarin to yield fluorescent, sulfenic acid-reactive compounds(6 and 7 in Scheme 3). Data presented herein confirm the utility ofthese reagents in specifically and rapidly labeling sulfenic acid groupsin proteins. Further, the synthetic methods should be generally usefulfor linking other types of fluorescent or affinity tags to sulfenicacid-containing proteins for analysis and isolation.

Synthesis. As preliminary biochemical results revealed that1,3-cyclohexadione reacts in a similar manner to dimedone, the protectedversion of 1,3-cyclohexadione, 3-ethoxy-2-cyclohexen-1-one (1, Scheme1), was chosen as the initial starting material for the preparation offluorescently-labeled probes. Starting with 1, which readily hydrolyzesto 1,3-cyclohexadione under acidic conditions,

removes any synthetic complications from the highly acidic(pK_(a)=5.15)(12) active methylene group of dimedone. Alkylation of theanion of commercially available 3-ethoxy-2-cyclohexen-1-one witht-butyldimethylsilyl (TBDMS)(13)-protected 3-iodo-1-propanol yields 2(51% yield, Scheme 1). Treatment of 2 with tetra-n-butylammoniumfluoride (TBAF) removes the silyl group to give the alcohol (3, 100%,Scheme 1). The alcohol group of 3 provides an attachment site for thefluorescent groups.

Condensation of 3 with N-methylisatoic anhydride yields the protected1,3-cyclohexadione derivative (4, 45%, Scheme 2). Heating7-methoxycoumarin-3-carboxylic acid in the presence of diphenylphosphorazidate (DPPA) and 3 gives the protected derivative (5, 50%,Scheme 2), presumably through the Curtius rearrangement of the acylazide to the isocyanate followed by condensation of 3. Treatment of 4and 5 with aqueous HC1 cleanly produces 3-(2,4-dioxocyclohexyl)propyl2-(methylamino)benzoate (6, DCP-MAB, Scheme 3) and3-(2,4-dioxocyclohexyl)propyl 7-methoxy-2-oxo-2H-chromen-3-ylcarbamate(7, DCP-MCC, Scheme 3) in 96 and 59% yield, respectively. Both the esterlinkage of 6 and the carbamate linkage of 7 appear generally stable tothese acidic deprotection conditions.

Reactivity of new compounds toward cysteine sulfenic acids in proteins.To test for the reactivity of 6 and 7 toward protein sulfenic acids, amutant, cysteine-dependent peroxidase enzyme was used in which theoxidized, sulfenic acid form of the cysteinyl redox center isstabilized, yet accessible (the C165S mutant of AhpC from Salmonellatyphimurium, a peroxiredoxin)(3, 9, 14). Both compounds, dissolvedinitially in dimethylsulfoxide and diluted 20-fold to a finalconcentration of 5 mM in aqueous, neutral pH buffer, gave covalentadducts with the sulfenic, but not sulfinic or sulfonic, acid forms ofthe peroxidase (Scheme 4).

The thiol group of the reduced protein and the oxidized,disulfide-bonded form of wild type AhpC were also unreactive towardthese compounds, as confirmed by electrospray ionization massspectrometry (ESI-MS) of the ammonium bicarbonate-washed

proteins following incubations with the labeling agents (FIG. 1 and datanot shown). As illustrated in FIG. 1 and summarized in Table 1, massspectrometry results were completely consistent with the expectedreactivity and products generated with the sulfenic acid-containingprotein using dimedone, 1,3-cyclohexadione and our two new labelingagents (6 and 7). Where sulfenic acid and hence adduct generation wassubstoichiometric due to the rapid addition of excess hydrogen peroxide(converting much of the enzyme to the sulfinic and perhaps sulfonic acidforms), additional peaks for thiol (unoxidized), and hyperoxidized(R—SO₂ ⁻ and R—SO₃ ⁻) protein were observed (FIG. 1). Under theconditions used, the sulfenic acid form of the protein either reactswith the reagent or is further oxidized during the workup or analysis ofthe sample, and is therefore not observed. In a subsequent experiment

TABLE 1 Observed and predicted masses of products following sulfenicacid-directed labeling of target protein (C165S AhpC). Observed increasein mass Predicted of product compared with R- additional mass Reagent SHform^(a) of product^(b) 1,3-cyclohexadione 109.2 110.1 DCP-MAB (6) 300.7301.4 DCP-MCC (7) 384.5 385.4 Dimedone 137.3 138.2 ^(a)Mass fromelectrospray ionization mass spectrometry on a Micromass Quattro IItriple quadrapole mass spectrometer of the modified product from whichwas subtracted the mass for the oxidized sulfinic acid product (R-SO₂ ⁻)present in each sample, adjusted 32 amu for the two oxygen atoms.^(b)Mass of labeling agent minus 2.016 for loss of hydrogens duringadduct formation.where sulfenic acid formation was measured at ˜73%, an equivalent amountof 6 or 7 (74 and 67%, respectively) was incorporated into the proteinas observed by ESI-MS (Table 1).

The fluorescence properties of 7 (λ_(ex,max)=341 nm and λ_(em,max)=414nm) were unchanged upon reaction with the cysteine sulfenic acid, whilesmall shifts in both the excitation and emission wavelength maxima wereobserved upon adduct formation with 6, the isatoic acid derivative (from2_(ex,max)=353 nm to λ_(ex,max)=357 nm, and from λ_(em, max)=440 nm toλ_(em,max)=430 nm, FIG. 2). Though small, these spectral changes allowone to directly and continuously monitor the fluorescence changes as 6reacts with the sulfenic acid-containing protein. In 45 mM potassiumphosphate buffer at pH 7 and 23° C., the second order rate constant forthe reaction of 6 with the R-SOH of the C165S mutant of AhpC is −500 M⁻¹min⁻¹ (data not shown). Protein adduct amounts assessed by ESI-MSanalyses of the intact proteins indicate approximately equal labeling ofthe protein with the two reagents when 1:1 mixtures of the 6 and 7 areadded, suggesting similar rates of reaction for these two compounds.While it is unlikely that all protein sulfenic acids react with thesecompounds at the same rate due in part to varying accessibility of thereactive group, it is probable that, under denaturing and anaerobicconditions, reaction rates will be similar to those with the modelprotein sulfenic acid.

Specificity of fluorescent, cyclohexadione-derived regents towardsulfenic acids in proteins. The known reactivity of the nucleophiliccenter of dimedone is toward cysteine sulfenic acids and aldehydes (15,16). Amines have also been shown to condense with dimedone (17). Asdescribed above, control reactions of thiol, disulfide or hyperoxidizedforms of AhpC (wild type or C165S) demonstrated their lack of reactivitytoward 6, 7 and dimedone based on the lack of ESI-MS-detectable adductformation. To test for general cross-reactivity of these reagents withother oxidized sulfur-containing functional groups, we tested thereactivity of dimedone, as a model reagent, with a S-nitrosothiol andtwo sulfoxides. Dimedone fails to react with S-nitrosoglutathione (GSNO)as judged by absorbance spectroscopy over one hour at room temperature.Nuclear magnetic resonance (NMR), spectroscopic and chemical isolationexperiments show that dimedone does not react with aqueous solutions ofeither dimethyl sulfoxide or methionine sulfoxide (data not shown).

As reported, dimedone demonstrates reactivity with both aldehydes andamines (16, 17). Control reactions show that dimedone reacts withbutyraldehyde in the presence of piperidine at 50° C. in aqueous ethanolbut fails to react with the same aldehyde at room temperature in theabsence of base. In addition, while dimedone condenses with benzylamineto form an imine in organic solvent, no reaction occurs in aqueousethanol. The failure of 6 and 7 to react with either reduced or oxidizedwild type or reduced C165S AhpC proteins also indicates that thesecompounds do not react with protein amine groups under these conditions.Taken together, these results demonstrate the relative specificity ofthe reaction of these compounds for sulfenic acids in proteins inaqueous buffers.

Conclusions. Functionalization of 1,3-cyclohexadione derivatives with analcohol group and subsequent coupling to fluorophores has successfullygenerated two sulfenic acid-reactive compounds that specificallyincorporate a fluorescent label into a sulfenic acid-containing modelprotein. Compounds such as these should prove useful in tagging proteinsulfenic acids for their detection and isolation from complex proteinmixtures in the future.

Experimental Section

3-Ethoxy-6-(3-t-butyldimethylsilyloxypropyl)cyclohex-2-enone (2). To alithium diisopropylamide (LDA) solution [prepared from diisopropylamine(3.82 mL, 27 mmol) and nBuLi (7.26 mL of a 2.5 M solution in hexanes, 18mmol) in tetrahydrofuran (THF, 12 mL) at 0° C.] at −78° C. was added3-ethoxy-2-cyclohexen-1-one (2.64 mL, 18 mmol) in THF (6 mL), dropwise,over 40 min. After stirring for an additional 30 min at −78° C.,hexamethyl phosphoramide (HMPA, 3.16 mL, 18 mmol) was added followed bythe dropwise addition of 3-iodo-1-tert-butyldimethylsiloxypropane (5.45g, 18 mmol) in THF (8 mL). The resultant mixture was allowed to warm tort, stirred for 6 h and then quenched by the addition of water (10 mL).The reaction mixture was then partitioned between dichloromethane (DCM,100 mL) and sat. NH₄Cl (40 mL). The aqueous phase was extracted with DCM(3×50 mL), the organic phases combined and washed with brine (50 mL),dried over anhydrous MgSO₄ and reduced to dryness. The resultant syrupwas purified by flash column chromatography (hexanes/EtOAc 8/2) to yield2 as a pale yellow oil (2.92 g, 51.2%). Rf 0.21 (hexanes/EtOAc 8/2);¹HNMR (300 MHz, CDCl₃) δ 5.26 (1H, s), 3.83 (2H, q, J=7.0 Hz), 3.59 (1H,t, J=6.5 Hz), 3.58 (H, t, J=6.5 Hz), 2.38 (2H, t, J=6.2 Hz), 2.20-1.99(2H, m), 1.85-1.35 (5H, in), 1.31 (3H, t, J=7.0 Hz), 0.84 (s, 9H), 0.00(s, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 201.6, 176.7, 102.3, 64.2, 63.4,45.0, 30.5, 28.0, 26.4, 26.0 (x2), 18.4, 14.2, -5.2.

3-Ethoxy-6-(3-hydroxypropyl)cyclohex-2-enone (3). To a solution of 2 inTHF (20 mL) was added TBAF (23.2 mL of a 1.0 M solution in THF, 23.2mmol) and NEt₃ (3.2 mL, 23.2 mmol). After stirring at rt for 2 h, thereaction was quenched by the addition of water (20 mL) and sat. NH₄Cl(20 mL). The mixture was extracted with DCM (3×80 mL) and the combinedorganic phases dried over anhydrous MgSO₄ and reduced to dryness. Theresultant syrup was purified by flash column chromatography withgradient elution (DCM/diethyl ether 1/1 to 3/7) to yield 3 as a paleyellow oil (1.85 g, 100%). Rf 0.37 (hexanes/EtOAc/MeOH 6/3/1); ¹H NMR(300 MHz, CDCl₃) δ 5.32 (1H, s), 3.89 (2H, q, J=7.1 Hz), 3.64 (2H, t,J=6.2 Hz), 2.44 (2H, 2d, J=7.1 Hz), 2.24 (1H, m), 2.06 (1H, m),1.92-1.49 (5H, m), 1.36 (3H, t, =7.1 Hz); ¹³C NMR (75 MHz, CDCl₃) δ202.0, 177.2, 102.3, 64.4, 62.6, 44.9, 30.3, 28.4, 26.8, 25.8, 14.3.

3-(4-Ethoxy-2-oxocyclohex-3-enyl)propyl 2-(methylamino)benzoate (4).Alcohol 3 (180 mg, 0.89 mmol), NEt₃ (0.56 mL) and catalytic DMAP wereadded to a solution of N-methylisatoic anhydride (220 mg, 0.89 mmol) indry DMF (2.0 mL) at rt. After stirring overnight, the solution waswarmed to 65° C. for 3 h. After cooling to rt, water was added and thismixture was extracted with ethyl acetate (5×50 mL). The combined organiclayers were washed with brine, dried over Na₂SO₄, filtered, andevaporated to give the crude product, which was purified by flash columnchromatography to afford 87.0 mg (45% yield) of 4 as a white solid: ¹HNMR (300MHz, CDCl₃) δ 7.85 (1H, in), 7.33 (1H, m), 6.56 (2H, m), 5.27(1H, s), 4.22 (2H, t, J=5.2 Hz), 3.85 (2H, q, J=7.0 Hz), 2.86 (3H, s),2.39 (2H, t, J=5.3 Hz), 2.04-1.41 (7H, in), 1.31 (3H, t, J=7.0 Hz); ¹³CNMR (75 MHz, CDCl₃) δ 201.1, 176.8, 168.7, 151.7, 134.7, 131.7, 115.0,111.3, 110.6, 102.5, 101.9, 64.7, 64.5, 64.3, 44.9, 30.0, 28.2, 26.6,26.5, 26.4, 14.3; ESI MS m/z 354 (M⁺+Na⁺).

3-(4-Ethoxy-2-oxocyclohex-3-enyl)propyl-7-methoxy-2-oxo-211-chromen-3-ylcarbamate (5). A solution of 7-methoxy-3-carboxycoumarin (335mg, 1.5 mmol), NEt₃ (1.05 mL, 7.6 mmol) and DPPA (0.36 mL, 1.7 mmol) inbenzene (15 mL) was stirred at 65° C. for 4 h. A solution of 3 (260 mg,1.3 mmol) in benzene (2 mL) was then added and the mixture stirred at65° C. for 16 h. Upon cooling, water (40 mL) was added and the mixtureextracted with DCM (3×40 mL). The combined organic phases were washedwith sat. NaHCO₃ (30 mL), brine (30 mL), dried over anhydrous MgSO₄ andreduced to dryness. The resultant solid was purified by flash columnchromatography (×2, gradient elution with DCM/EtOAC (8/2) toDCM/EtOAc/MeOH (8/1/1), and then DCM/MeOH (9/1)) to yield 5 as anoff-white solid (270 mg, 49.5%). Rf 0.63 (hexanes/EtOAc 1/2); Mp.131-134° C.; ¹H NMR (300 MHz, CDCl₃) δ 8.22 (1H, s), 7.34 (2H, m), 6.84(1H, dd, J=8.6 Hz, 2.4 Hz), 6.79 (1H, d, J=2.4 Hz), 5.29 (1H, s), 4.18(2H, t, J=6.3 Hz), 3.85 (2H, 2q, J=7.0 Hz), 3.82 (3H, s), 2.42 (2H, 2d,J=7.1 Hz), 2.22 (1H, m), 2.08 (1H, dq, J=14.2 Hz, 5.0 Hz), 1.94-1.85(1H, m), 1.78-1.66 (3H, m), 1.52-1.42 (1H, m), 1.33 (3H, t, J=7.0 Hz);¹³C NMR (75 MHz, CDCl₃) δ 201.0, 176.9, 161.1, 158.9, 153.6, 151.3,128.4, 122.0, 121.9, 113.3, 113.2, 102.4, 100.8, 66.0, 64.4, 55.9, 44.9,28.3, 26.6 (×2), 26.2, 14.3; ESI MS m/z 438 (M⁺+Na⁺).

3-(2,4-Dioxocyclohexyl)propyl 2-(methylamino)benzoate (DCP-MAB, compound6). Compound 4 was stirred in a mixture of THF/3N HCl (1/1) at rt for 3h and the mixture was concentrated. The crude product was washed withEtOAc/hexane/MeOH (1/2/0.5) to afford 6 as a white solid: ¹H NMR (300MHz, CDCI₃) δ 7.83 (1H, m), 7.34 (1H, m), 6.55 (2H, m), 4.22 (2H, t,J=6.3 Hz), 3.60 (2H, m), 2.84 (3H, s), 2.52-1.50 (9H, m); ¹³C NMR(75MHz, CDCl₃) δ 204.3, 203.8, 168.6, 151.6, 134.9, 131.7, 131.5, 115.2,111.6, 110.5, 64.1, 58.4, 49.1, 39.9, 30.1, 26.4, 26.0, 24.7; ESI MS m/z326 (M⁺+Na⁺).

3-(2,4-Dioxocyclohexyl)propyl 7-methoxy-2-oxo-2H-chromen-3-ylcarbamate(DCP-MCC, compound 7). Compound 5 (240 mg, 0.58 mmol) was stirred in amixture of THF/DCM (3/1 v/v, 4 mL) and 3N HCl (4 mL) for 1 hr. Thereaction mixture was diluted with water (10 mL) and extracted with DCM(3×30 mL). The combined organic phases were dried over anhydrous MgSO₄and reduced to dryness to yield the crude product, which was purified byflash column chromatography (EtOAc/acetone 4/1) to yield 7 as anoff-white solid (133 mg, 59.4%). Rf 0.62 (hexanes/EtOAc/MeOH 6/3/1); Mp.154-156° C.; ¹H NMR (300 MHz, CDCl₃) δ 8.25 (1H, s), 7.42 (1H, s), 7.39(1H, d, J=8.7 Hz), 6.88 (1H, dd, J=8.6 Hz, 2.1 Hz), 6.83 (1H, d, J=2.1Hz), 4.23 (2H, t, J=6.5 Hz), 3.86 (3H, s), 3.44 (2H, d, J=5.1 Hz),2.78-2.69 (1H, dt, J=16.4 Hz, 4.4 Hz), 2.67-2.51 (2H, m), 2.24-2.15 (1H,dq, J=14.1 Hz, 4.8 Hz), 2.03-1.95 (1H, m), 1.88-1.47 (6H, m); ¹³C NMR(75 MHz, CDCl₃) δ 204.2, 203.8, 161.2, 158.9, 153.5, 151.3, 128.4,122.0, 121.8, 113.3, 113.2, 100.9, 65.6, 58.5, 55.9, 49.1, 39.9, 26.6,25.7, 24.8; ESI MS m/z 410 (M⁺+Na⁺), 388 (M⁺+H⁺); CHN calc. forC₂₀H₂₁NO₇.0.5H₂O, C: 60.59, H: 5.61, N: 3.53, found C: 61.31, H: 5.56,N: 3.48.

Generation of pure, sulfenic acid-containing C165S AhpC. The C165Smutant of AhpC was expressed in bacteria and purified in the presence of5 mM 1,4-dithiothreitol (added to all buffers) essentially as describedpreviously (9). Purification of wild type AhpC was also carried out aspreviously described (10). For generation of the sulfenic acid form ofC165S AhpC, thawed enzyme in 25 mM phosphate buffer at pH 7, containing1 mM EDTA, was washed free of the dithiothreitol and transferred intofresh buffer using a PD-10 column (Amersham Biosciences), and pooledfractions were transferred to an anerobic cuvette. The protein solution(160 nmol in 0.60 mL) was made anaerobic by repeated cycles of argon andvacuum over about 30 min, then rapidly titrated with 11 mM hydrogenperoxide to give a maximal absorbance signal at ˜367 nm for thesulfenate anion as described previously (3, 4). In subsequentexperiments, optimal sulfenic acid formation was observed by adding andmixing the hydrogen peroxide much more slowly (additions of about 0.05eq and thorough mixing every ˜1 min) and quantitating the sulfenic acidformed using a freshly-prepared 2-nitro-5-thiobenzoate (TNB) solution(0.28 mL) and a 60 μL aliquot of the protein removed anaerobically fromthe cuvette (3, 4). Using this method, ˜73% of the protein was convertedto the sulfenic acid form after addition of 1.08 eq of hydrogenperoxide. Protein was kept in the anaerobic cuvette at room temperaturefor several hours before conducting experiments with no loss in sulfenicacid content.

Labeling of sulfenic acid-containing C165S AhpC for spectral and massspectrometric analysis. The pure, oxidized enzyme (32 μL, 6 nmol each)from the anaerobic cuvette was added to argon-flushed 500 μL Eppendorftubes containing the compound of choice (either dimethylsulfoxide alone,or dimethylsulfoxide into which dimedone, 1,3-cyclohexanedione, 6 or 7had been dissolved) to give a final concentration of 5 mM for the addedreagent in final volumes of 50 μL, and the tubes were flushed again withargon before closing. Other redox forms of C165S AhpC or the wild-typeenzyme were also tested for reactivity with one or more of these fourcompounds. Approximate 100 mM stock solutions of 6 or 7 indimethylsulfoxicle were standardized using expected extinctioncoefficients of 5,700 or 25,000 M⁻¹ cm⁻¹, respectively, for theesterified isatoic acid conjugate or the methoxycoumarin conjugate inmethanol (11). Samples were incubated at room temperature for 60 min,then transferred to Apollo ultrafiltration devices (30K cutoff, OrbitalBiosciences, Topsfield, Mass.), washed with 5.5 mL 10 mM ammoniumbicarbonate and reconcentrated to about 50 μL, for a total of fourwashes (>10⁸-fold dilution of the initial small molecule components).

Mass spectrometric and spectral analyses of adducts with the sulfenicacid-containing form of C165S AhpC. For mass spectrometric analyses, 60μL of the ammonium bicarbonate buffer containing 1-2 nmol of labeled orunlabeled protein was submitted to the Mass Spectrometer Facility atWake Forest University School of Medicine for infusion analysis on aMicromass Quattro II triple quadrupole mass spectrometer equipped with aZ-spray source. Just prior to analysis, samples were diluted 1:1 withacetonitrile and 1% formic acid was added. The data were processed andanalyzed using MassLynx Version 3.5.

Labeled protein samples washed (with Apollo concentrators) intopotassium phosphate or ammonium bicarbonate buffers were also analyzedfor their UV-visible spectroscopic and fluorescence properties using aBeckman DU7500 diode array spectrophotometer or a SLM Aminco-BowmanSeries 2 luminescence spectrophotometer, respectively. By comparisonwith the reported extinction coefficients for each of the free reagentsin methanol (5,700 M⁻¹ cm⁻¹ for esterified isatoic acid, and 25,000 M⁻¹cm⁻¹ for methoxycoumarin) (11), the free and presumably protein-boundreagents in neutral pH phosphate buffer exhibited average extinctioncoefficients of ˜4,250 and ˜20,900 M⁻¹ cm⁻¹, respectively, for 6 and 7.These measured values were subsequently used to standardize 6 and 7 andto estimate the labeled protein concentrations.

Analysis of the rate of reaction of the cysteine sulfenicacid-containing C165S AhpC with DCP-MAB (6), and of other potentiallyreactive compounds with dimedone. To 10 nmol of sulfenic acid-containingC165S AhpC in 50 mM potassium phosphate buffer at pH 7.0 was added 6(from a 119 mM stock solution in dimethyl sulfoxide) at finalconcentrations of 0.6 to 2.4 mM and a total volume of 0.5 mL.Fluorescence descreases were monitored at 23° C. using excitation andemission wavelengths of 357 and 464 nm and slit widths of 4 and 16 nm,respectively. Both free and protein-bound DCP-MAB exhibit the sameabsorbance at this excitation wavelength, allowing the experiment to beconducted at high concentrations without complications due to the innerfilter effect (A₃₅₇ of ˜2 to 10). Fluorescence changes were monitored at15 s to 2 min intervals until complete, and each first order rateconstant was analyzed using Kaleidagraph (Synergy Software) and theequation:

y=A+Be ^(−k) ^(obs) ^(t)

where y is the fluorescence value at a given time t, and A and B are thefinal fluorescence value (A) and amplitude of fluorescence change (B)parameters associated with the change, and k_(obs) is the pseudo-firstorder rate constant (with A, B and k_(obs) supplied by the fit to thedata). The slope of the secondary plot of the observed rate constant,k_(obs), versus concentration of 6 was taken as the second order rateconstant for the reaction of 6 with the cysteine sulfenicacid-containing C165S mutant of AhpC.

In addition to the compounds described above, several additionalcompounds have been synthesized with fluorophore or biotin labels asdescribed below.

Synthesis of a 1,3-cyclohexadione-fluorescein derivative. Condensationof commercial fluoresceinamine with p-nitrophenyl chloroformate gives areactive carbamate that condenses with alcohol (3, Scheme 1) to producea protected 1,3-cyclohexadione-fluorescein derivative (Scheme 5). Acidicdeprotection of the enol ether group yields the desired probe in goodyield (Scheme 5).

Synthesis of a 1,3-cyclohexadione-biotin derivative. Standard couplingof commercial biotin using dicyclocarbodiimide (DCC) with alcohol (3,Scheme 1) gives a protected 1,3-cyclohexadione-biotin derivative (Scheme6). Further acidic deprotection of the enol ether group yields thedesired biotin-based probe in good yield (Scheme 6).

REFERENCES

-   1. Claiborne, A., Yeh, J. I., Mallett, T. C., Luba, J., Crane, E.    J., 3rd, Charrier, V., and Parsonage, D. (1999) Protein-sulfenic    acids: diverse roles for an unlikely player in enzyme catalysis and    redox regulation, Biochemistry 38, 15407-15416.-   2. Poole, L. B., Karplus, P. A., and Claiborne, A. (2004) Protein    sulfenic acids in redox signaling, Annu. Rev. Pharmacol. Toxicol.    44, 325-347.-   3. Poole, L. B., and Ellis, H. R. (2002) Identification of cysteine    sulfenic acid in AhpC of alkyl hydroperoxide reductase, Methods    Enzymol 348, 122-136.-   4. Poole, L. B. (2003) Measurement of protein sulfenic acid content,    in Curr. Prot. Toxicol. (Maines, M. D., Ed.) pp 17.12.11-17.12.20,    John Wiley & Sons, Inc., New York.-   5. Allison, W. S. (1976) Formation and reactions of sulfenic acids    in proteins, Acc. Chem. Res. 9, 293-299.-   6. Willett, W. S., and Copley, S. D. (1996) Identification and    localization of a stable sulfenic acid in peroxide-treated    tetrachlorohydroquinone dehalogenase using electrospray mass    spectrometry, Chem Biol 3, 851-857.-   7. Detection of radioactively-labeled dimedone has also been used in    the past, although this material is no longer commercially available    and is prohibitively expensive to synthesize.-   8. Saurin, A. T., Neubert, H., Brennan, J. P., and Eaton, P. (2004)    Widespread sulfenic acid formation in tissues in response to    hydrogen peroxide, Proc Natl Acad Sci USA 101, 17982-17987.-   9. Ellis, H. R., and Poole, L. B. (1997) Roles for the two cysteine    residues of AhpC in catalysis of peroxide reduction by alkyl    hydroperoxide reductase from Salmonella typhimurium, Biochemistry    36, 13349-13356.-   10. Poole, L. B., and Ellis, H. R. (1996) Flavin-dependent alkyl    hydroperoxide reductase from Salmonella typhimurium. 1. Purification    and enzymatic activities of overexpressed AhpF and AhpC proteins,    Biochemistry 35, 56-64.-   11. Haugland, R. P. (2002) Handbook of fluorescent probes and    research products, Ninth ed., Molecular Probes, Inc.-   12. The Merck Index, 12th Edition; Merck and Co., Inc: Whitehouse    Station, 1996, p 548.-   13. Abbreviations used are: TBDMS, t-butyldimethylsilyl; TBAF,    tetra-n-butylammonium fluoride; amu, atomic mass units; THF,    tetrahydrofu ran; HMPA, hexamethylphosphoramide; DMAP, dimethyl    amino pyridine; DPPA, diphenyl phosphorazidate; GSNO,    S-nitrosoglutathione; NMR, nuclear magnetic resonance; LDA, lithium    diisopropylamide; DCM, dichloromethane; ESI-MS, electrospray    ionization mass spectrometry; NBD chloride,    7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; DCP-MAB,    3-(2,4-dioxocyclohexyl)propyl 2-(methylamino)benzoate;DCP-MCC,    3-(2,4-dioxocyclohexyl)propyl    7-methoxy-2-oxo-2H-chromen-3-ylcarbamate-   14. Ellis, H. R., and Poole, L. B. (1997) Novel application of    7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine    sulfenic acid in the AhpC component of alkyl hydroperoxide    reductase, Biochemistry 36, 15013-15018.-   15. Benitez, L. V., and Allison, W. S. (1974) The inactivation of    the acyl phosphatase activity catalyzed by the sulfenic acid form of    glyceraldehyde 3-phosphate dehydrogenase by dimedone and olefins, J    Biol Chem 249, 6234-6243.-   16. Vogel, A. I. (2005) Investigation and characterisation of    organic compounds, in Vogel's Textbook of Practical Organic    Chemistry (Furniss, B. S., Hannaford, A. J., Smith, P. W. G., and    Tatchell, A. R., Eds.) pp 1259-1260, Pearson, Singapore.-   17. Halpern, B., and James, L. B. (1964) Dimedone (5,    5-dimethylcyclohexane-1, 3-dione) as a protecting agent for amino    groups in peptide synthesis, Aust. J. Chem. 17, 1282-1287.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-7. (canceled)
 8. A method of synthesizing a compound of Formula I:

wherein: R₁ is a label selected from the group consisting of detectablegroups and anti-tumor agents; L is present or absent and when present isa linking group; and x represents an integer from 1 to 10, comprising:reacting 3-ethoxy-2-cyclohexen-1-one with a t-butyldimethylsilyl (TBDMS)protected halo lower alkyl alcohol in hexamethylphosphoramide to form a6-lower alkyl-OtBDMS-3-ethoxy-2-cyclohexen-1-one solution, adding tetrabutyl ammonium fluoride (TBAF) to said solution to form an alcoholsolution, adding a label to the alcohol solution, and adding HCl tocreate a ketone at the (C)3 position.
 9. The method of claim 8, whereinthe label is selected from the group consisting of biotin, fluorophores,antigens, porphyrins, radioactive isotopes and antitumor agents.
 10. Themethod of claim 8, wherein said label is a fluorophore.
 11. The methodof claim 8, wherein the label is a fluorophore selected from the groupconsisting of 1-methyl-2H-3,1-benzoxazine-2,4(1H)-dione (N-Methylisatoicanhydride) and a composition of diphenyl phosphorazidate (DPPA),7-methoxycoumarin-3-carboxylic acid, and fluorescein.
 12. The method ofclaim 8, wherein the lower alkyl alcohol is 3-iodo-1-propanol.
 14. Amethod of identifying sulfenic acids in a protein, comprising: applyinga compound of Formula I:

wherein: R₁ is a label selected from the group consisting of detectablegroups and anti-tumor agents; L is present or absent and when present isa linking group; and x represents an integer from 1 to 10, to saidprotein, and then detecting the presence or absence of binding of saidcompound to said protein, the presence of binding indicating thepresence of sulfenic acids in said protein.
 15. The method of claim 14,wherein said label is selected from the group consisting of biotin,fluorophores, antigens, porphyrins, and radioactive isotopes.
 16. Themethod of claim 14, wherein said label is a fluorophore.
 17. A methodfor monitoring oxidative damage in proteins or cells comprising:reacting compound of claim 1 with a protein comprising cysteineresidues, a mixture of proteins, a cell lysate, or intact orpermeabilized cells; wherein an increase in binding of said compound tosaid proteins or cells as compared to corresponding control proteins orcells prior to said oxidative damage indicates oxidative damage to saidproteins or cells.
 18. The method of claim 17, wherein the label isselected from the group consisting of biotin, fluorophores, antigens,porphyrins, and radioactive isotopes.
 19. The method of claim 17,wherein said label is a fluorophore. 20-23. (canceled)
 24. The method ofclaim 8, wherein said compound of Formula I is selected from the groupconsisting of: (a) compounds of formula II:

wherein x represents an integer from 1 to 10; and (b) compounds offormula III:

wherein x represents an integer from 1 to
 10. 25. The method of claim14, wherein said compound of Formula I is selected from the groupconsisting of: (a) compounds of formula II:

wherein x represents an integer from 1 to 10; and (b) compounds offormula III:

wherein x represents an integer from 1 to 10.